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Reduction of cytochrome c oxidase by a second electron leads to proton translocation


Cytochrome c oxidase, the terminal enzyme of cellular respiration in mitochondria and many bacteria, reduces O2 to water. This four-electron reduction process is coupled to translocation (pumping) of four protons across the mitochondrial or bacterial membrane1; however, proton pumping is poorly understood. Proton pumping was thought to be linked exclusively to the oxidative phase, that is, to the transfer of the third and fourth electron2. Upon re-evaluation of these data, however, this proposal has been questioned3,4, and a transport mechanism including proton pumping in the reductive phase—that is, during the transfer of the first two electrons—was suggested. Subsequently, additional studies reported that proton pumping during the reductive phase can occur, but only when it is immediately preceded by an oxidative phase5. To help clarify the issue we have measured the generation of the electric potential across the membrane, starting from a defined one-electron reduced state. Here we show that a second electron transfer into the enzyme leads to charge translocation corresponding to pumping of one proton without necessity for a preceding turnover.

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Figure 1: Outline of the catalytic cycle and the structure of cytochrome c oxidase.
Figure 2: Absorbance difference spectra demonstrating the generation and decay of the one-electron reduced state E.
Figure 3: Membrane potential on injection of single electrons into cytochrome c oxidase.
Figure 4: Comparison of photopotentials upon photolysis of state MV-CO and upon successive electron injections into state E.


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We thank the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie and the Max-Planck-Gesellschaft for financial support. We are grateful to B. Ludwig for providing the D124N mutant.

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Correspondence to Klaus Fendler.

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Ruitenberg, M., Kannt, A., Bamberg, E. et al. Reduction of cytochrome c oxidase by a second electron leads to proton translocation. Nature 417, 99–102 (2002).

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